DNA polymerase beta: contributions of template-positioning and dNTP triphosphate-binding residues to catalysis and fidelity.

The specific catalytic roles of two groups of DNA polymerase beta active site residues identified from crystal structures were investigated: residues possibly involved in DNA template positioning (Lys280, Asn294, and Glu295) and residues possibly involved in binding the triphosphate moiety of the incoming dNTP (Arg149, Ser180, Arg183, and Ser188). Eight site-specific mutants were constructed: K280A, N294A, N294Q, E295A, R149A, S180A, R183A, and S188A. Two-dimensional NMR analysis was employed to show that the global conformation of the mutants has not been perturbed significantly. Pre-steady-state kinetic analyses with single-nucleotide gapped DNA substrates were then performed to obtain the rate of catalysis at saturating dNTP (k(pol)), the apparent dissociation constant for dNTP (K(d)), catalytic efficiency k(pol)/K(d), and fidelity. Of the three template-positioning residues, Asn294 and Glu295 (but not Lys280) contribute significantly to k(pol). Taken together with other data, the results suggest that these two residues help to stabilize the transition state during catalysis even though they interact with the DNA template backbone rather than directly with the incoming dNTP or the opposite base on the template. Furthermore, the fidelity increases by up to 19-fold for N294Q due to differential k(pol) effects between correct and incorrect nucleotides. Of the four potential triphosphate-binding residues, Ser180 and Arg183 contribute significantly to k(pol) while the effects of R149A are relatively small and are primarily on K(d), and Ser188 appears to play a minimal role in the catalysis by Pol beta. These results identify several residues important for catalysis and quantitate the contributions of each of those residues. The functional data are discussed in relation to the prediction on the basis of available crystal structures.

Localized Rac activation dynamics visualized in living cells.

Signaling proteins are thought to be tightly regulated spatially and temporally in order to generate specific and localized effects. For Rac and other small guanosine triphosphatases, binding to guanosine triphosphate leads to interaction with downstream targets and regulates subcellular localization. A method called FLAIR (fluorescence activation indicator for Rho proteins) was developed to quantify the spatio-temporal dynamics of the Rac1 nucleotide state in living cells. FLAIR revealed precise spatial control of growth factor-induced Rac activation, in membrane ruffles and in a gradient of activation at the leading edge of motile cells. FLAIR exemplifies a generally applicable approach for examining spatio-temporal control of protein activity.

DNA polymerase beta: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes.

Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.

DNA polymerase beta: analysis of the contributions of tyrosine-271 and asparagine-279 to substrate specificity and fidelity of DNA replication by pre-steady-state kinetics.

DNA polymerase beta (pol beta) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd(app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol beta, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol beta. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol beta. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol beta. Since the chemical step may not be rate-limiting for wild-type pol beta, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol beta displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.

DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity.

DNA polymerase beta (pol beta) is the smallest and least complex DNA polymerase. The structure of the enzyme is well understood, but little is known about its catalytic properties, particularly processivity and fidelity. Pre-steady-state analysis of the incorporation of a single nucleotide into a short 25/45 oligonucleotide primer-template by pol beta was used to define the kinetic parameters of the polymerase. In addition, nucleotide analogs and site-specific mutants, along with structural analyses, were used to probe the structure-function relationship of pol beta. Several significant findings have been obtained: (i) The catalysis by pol beta is processive and displays an initial burst under pre-steady-state conditions, but the processivity is poor compared to other polymerases. (ii) The fidelity of pol beta is also low relative to other polymerases. (iii) Under pre-steady-state conditions the chemical step appears to be only partially rate-limiting on the basis of the low thio effect (4.3), defined as kpol(dNTP)/kpol(dNTP alpha S). The thio effect increases to 9 for incorporation of an incorrect nucleotide. These results are consistent with the existence of a substrate-induced conformational change that is also partially rate-limiting. (iv) A comparison between the two-dimensional NMR spectra of the wild-type and mutant enzymes indicates that the mutations at position 283 did not significantly perturb the structure of the enzyme. The conformational stability of the mutants is also unperturbed. Thus, R283 is not important to the overall structure of the enzyme. (v) The results of kinetic analyses of R283A and R283K mutants indicate that the hydrogen bond between R283 of pol beta and the template is important for catalysis. Both R283A and R283K mutants displayed decreases in catalytic efficiency by a factor of ca. 200 relative to wild-type pol beta. The mutants are also less faithful by a factor of 2-4, in terms of the T-G mispair vs the T-A correct pair. The perturbation, however, could occur at both the implied conformational step and the chemical step, since the thio effects of the mutants for both correct and incorrect nucleotides are similar to those of WT pol beta.